High-throughput detection of low abundance sialylated glycoproteins in human serum by TiO2 enrichment and targeted LC-MS/MS analysis: application to a prostate cancer sample set
Glycopeptide enrichment can be a strategy to allow the detection of peptides belonging to low abundance proteins in complex matrixes such as blood serum or plasma. Though several glycopeptide enrichment protocols have shown excellent sensitivities in this respect, few reports have demonstrated the applicability of these methods to relatively large sample cohorts. In this work, a fast protocol based on TiO2 enrichment and highly sensitive mass spectrometric analysis by Selected Reaction Monitoring (SRM) has been applied to a cohort of serum samples from prostate cancer and benign prostatic hyperplasia patients in order to detect low abundance proteins in a single LC-MS/MS analysis in nanoscale format, without immunodepletion or peptide fractionation. A peptide library of over 700 formerly N-glycosylated peptides was created by data dependent analysis. Then, 16 medium to low abundance proteins were selected for detection by single injection LC-MS/MS based on selected-reaction monitoring. Results demonstrated the consistent detection of the low-level proteins under investigation. Following label-free quantification, four proteins (Adipocyte plasma membrane-associated protein, Periostin, Cathepsin D and Lysosome-associated membrane glycoprotein 2) were found significantly increased in prostate cancer sera compared to the control group.
KeywordsProstate cancer Serum proteomics APMAP CTSD LAMP2 POSTN
MIUR, Programma Operativo Nazionale PON03PE_0009_2 “ICaRe”; POR Calabria FESR 2014-2020 “Innoprost”.
Compliance with ethical standards
Written informed consent was obtained from the patient for research use. The study was approved by the University of Catanzaro ethics committee.
Conflict of interest
The authors declare that they have no conflict of interest.
- 5.Liu M-Q, Zeng W-F, Fang P, Cao W-Q, Liu C, Yan G-Q, et al. pGlyco 2.0 enables precision N-glycoproteomics with comprehensive quality control and one-step mass spectrometry for intact glycopeptide identification. Nat Commun. 2017;8:438. https://doi.org/10.1038/s41467-017-00535-2.CrossRefGoogle Scholar
- 7.Lakbub JC, Su X, Zhu Z, Patabandige MW, Hua D, Go EP, et al. Two new tools for glycopeptide analysis researchers: a glycopeptide decoy generator and a large data set of assigned CID spectra of glycopeptides. J Proteome Res. 2017;16:3002–8. https://doi.org/10.1021/acs.jproteome.7b00289.CrossRefGoogle Scholar
- 13.Shah AK, Cao K-AL, Choi E, Chen D, Gautier B, Nancarrow D, et al. Serum glycoprotein biomarker discovery and qualification pipeline reveals novel diagnostic biomarker candidates for esophageal adenocarcinoma. Mol Cell Proteomics. 2015;14:3023–39. https://doi.org/10.1074/mcp.M115.050922.CrossRefGoogle Scholar
- 17.Zhao X, Ma C, Han H, Jiang J, Tian F, Wang J, et al. Comparison and optimization of strategies for a more profound profiling of the sialylated N-glycoproteomics in human plasma using metal oxide enrichment. Anal Bioanal Chem. 2013;405:5519–29. https://doi.org/10.1007/s00216-013-6971-5.CrossRefGoogle Scholar
- 19.Totten SM, Adusumilli R, Kullolli M, Tanimoto C, Brooks JD, Mallick P, et al. Multi-lectin affinity chromatography and quantitative proteomic analysis reveal differential glycoform levels between prostate cancer and benign prostatic hyperplasia sera. Sci Rep. 2018;8:6509. https://doi.org/10.1038/s41598-018-24270-w.CrossRefGoogle Scholar
- 20.Kälin M, Cima I, Schiess R, Fankhauser N, Powles T, Wild P, et al. Novel prognostic markers in the serum of patients with castration-resistant prostate cancer derived from quantitative analysis of the Pten conditional knockout mouse proteome. Eur Urol. 2011;60:1235–43. https://doi.org/10.1016/j.eururo.2011.06.038.CrossRefGoogle Scholar
- 21.Liu Y, Chen J, Sethi A, Li QK, Chen L, Collins B, et al. Glycoproteomic analysis of prostate cancer tissues by SWATH mass spectrometry discovers N-acylethanolamine acid amidase and protein tyrosine kinase 7 as signatures for tumor aggressiveness. Mol Cell Proteomics. 2014;13:1753–68. https://doi.org/10.1074/mcp.M114.038273.CrossRefGoogle Scholar
- 25.Taverna D, Mignogna C, Gabriele C, Santise G, Donato G, Cuda G, et al. An optimized procedure for on-tissue localized protein digestion and quantification using hydrogel discs and isobaric mass tags: analysis of cardiac myxoma. Anal Bioanal Chem. 2017;409:2919–30. https://doi.org/10.1007/s00216-017-0237-6.CrossRefGoogle Scholar
- 33.Saitoh O, Wang WC, Lotan R, Fukuda M. Differential glycosylation and cell surface expression of lysosomal membrane glycoproteins in sublines of a human colon cancer exhibiting distinct metastatic potentials. J Biol Chem. 1992;267:5700–11.Google Scholar